Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)

Definitions

• This invention provides an improved method for purification of recombinant copper/zinc (Cu-Zn) superoxide dismutase from bacteria or eucaryotic cells.
• SOD superoxide dismutase
• the superoxide anion (0o 2 ) produced on reperfusion of ischemic tissue and also in certain inflammatory conditions is highly toxic to macromolecules, e.g., 0o 2 may react with lipid hydroperoxides to form alkoxy radicals in phospholipid membranes.
• SOD SOD which increases the rate of inter ⁇ cellular dismutation by a factor of 10 9 .
• SOD is a ubiquitous mammalian enzyme. In the presence of normal intercellular concentrations of catalase and peroxidase, SOD is responsible for the "scavenging" of oxygen-free radicals, thereby serving as a normal biolog ical defense against the formation and accumulation of reduced oxygen intermediates.
• Copper/zinc (Cu-Zn) SOD has been demonstrated in virtually all eucaryotic organisms.
• Human Cu-Zn SOD-1 is a dimeric metallo-protein composed of identical non-co- valently linked subunits, each having a molecular weight of 16,000 daltons and containing one atom of copper and one of zinc. Each subunit is composed of 153 amino acids whose sequence has been established.
• Endogenous SOD is present in tissues in limited amounts and when high levels of superoxide anion are produced, the amount of SOD present is not sufficient. Thus there is a need for clinical administration of exogenous SOD.
• exploration of the therapeutic potential of human SOD-1 (EC 1.15.1.1) has been limited mainly due to its scarce availability.
• an SOD clone containing the entire coding region of human SOD-1 described by Groner and his colleagues (15) into efficient bacterial expression vectors which express hSOD analog at high levels; this has been described in coassigned U.S. Patent No. 4,742,004 described above.
• hSOD analog which differs from authentic human SOD (from blood) in that the amino terminus alanine is not acetylated.
• the amino acid sequence of the bacterial-produced SOD analog does not contain a methionine residue at its N- terminus.
• the recombinant hSOD analog thus produced is very pure. However, it was desirable to produce even higher levels of purity.
• An improved chromatography method which produces an even higher degree of purification. surprisingly achieved without loss of yield, is described in this application.
• a novel "exchange" procedure which increases the yield of pure recombinant SOD produced, without loss of purity, is also described.
• European patent application publication no. 0180964 published May 14, 1986 and assigned to Ube Industries Limited, discloses the production of human Cu-Zn SOD in Escherichia coli and partial purification of the hSOD produced. This method was done on small scale only (20 g of wet cells). No details of purification achieved are given apart from specific activity. There is no disclosure of the extent of contamination by Escherichia coli proteins and endotoxins.
• European patent application publication no. 0138111 published April 24, 1985 and assigned to Chiron Corporation, discloses the production of hSOD in both Escherichia coli and yeast, but no methods of production of hSOD from crude cell lysates are disclosed.
• Hallewell et al. from Chiron Corporation (16) disclose production of recombinant SOD but merely state that the recombinant SOD was purified to homogeneity by conventional means after lysing the yeast cells with glass beads and pelleting the cell debris.
• Hallewell et al. from Chiron Corporation (17) disclose a method of purifying recombinant SOD from yeast cells. This method gives no detail of the yield of hSOD produced, nor its degree of purification; it is also a purification method of hSOD analog from yeast cells and not Escherichia coli cells.
• Takahara et al. disclose the secretion by Escherichia coli cells of hSOD into the periplasmic space. This is a very small-scale procedure (16 ml medium), and the SOD produced is not purified; the Escherichia coli cells are simply subjected to osmotic shock, and subcellular fractions are analyzed by SDS- PAGE.
• the subject invention provides a method for recovering a solution containing purified, enzymatically active Cu-Zn superoxide dismutase or a polypeptide analog thereof having substantially the same amino acid sequence as, and the biological activity of, naturally- occurring Cu-Zn superoxide dismutase from a composition which comprises cells containing Cu-Zn superoxide dismutase or a polypeptide analog thereof comprising:
• plasmid pSOD ⁇ 1 T11 has been fully described in co-assigned, copending European patent application publication no. 0173280, published on May 5, 1986 (corresponding to U.S. patent application Serial No. 644,105, filed August 27, 1984, issued May 2, 1988 as U.S. Patent No. 4,742,004), and pSOD ⁇ 1 T11 has been deposited in the ATCC under Accession No. 53468.
• This plasmid contains the following elements:
• Plasmid pSOD ⁇ 1 T11 is a high level expressor of hSOD analog protein under the control of the strong leftward promoter of bacteriophage ⁇ (P L ) which is thermoin- ducibly controlled by the cI857 temperature sensitive repressor situated on the host chromosome.
• P L strong leftward promoter of bacteriophage ⁇
• This column may be used in the ion-exchange chromatography step of the method for purifying hSOD analogs.
• Application of hSOD analog solution to the DEAE-Sepharose FF column and elution conditions on the column are as described in the text (Example 3E).
• the CM-Sepharose FF column may also be used in the ionexchange chromatography step of the method for purifying hSOD analog.
• Application of the hSOD analog solution to the CM-Sepharose FF columns and solution conditions on the column are as described in the text (Example 3F).
• the grey peaks CW2, CE1 and CE2 can be collected and treated as described in Example 4B.
• B isolated isoform c of hSOD analog (2.5 mg/ml) .
• C mixture (1:1) of isolated isoforms a (5 mg/ml) and c (5 mg/ml) without incubation.
• the samples were dissolved in buffer A (25 mM bis-Tris- HC1, pH 7.0) and 100 ⁇ l of the solution was injected onto an analytical fast performance Mono Q (HR 5/5) column.
• Isoform b was preparatively isolated from a mixture of isolated isoforms a and c, following incubation for 24 hours at 37°C, by preparative fast performance chromatography on Mono Q (HR 10/10) column.
• the isoform b peak eluted was collected and immediately frozen at -20°C.
• the samples were defrosted and diluted 1:3 with H 2 O (yielding approximately 0.45 mg/ml).
• 200 ⁇ l of the solution was injected onto an analytical fast performance anion exchange Mono Q (HR 5/5) column. The elution was performed as described in Figure 3, except the detection at 280 nm was 0.05 AUFS.
• A the mixture was injected immediately after the relative fraction mixing.
• B the mixture was injected after incubation for two hours at 37oC and 20 hours at room temperature.
• the peak shaded black (DMP fraction) was collected and dialyzed against 40 mM sodium acetate pH 4.8.
• Sepharose column Application and elution conditions are the same as Figure 2.
• A a sample from DMP fraction derived from DEAE- Sepharose column (black shaded peak in Figure 7A) was diluted 1:4 in 20 mM bis Tris-HCl, pH 7.0, and 400 ⁇ l were injected onto a Mono Q (HR 5/5) column.
• the plasmids designated pSOD ⁇ 1 T11 , pSOD ⁇ MAX-12 , pSOD ⁇ 2, pMF2005, pMF5534, and pNd-SOD NN -12 were deposited pursuant to, and in satisfaction of, the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 53468, 67177, 39786, 67362, 67703, and 53166, respectively.
• ATCC American Type Culture Collection
• the subject invention provides a method for recovering a solution containing purified, enzymatically active Cu-Zn superoxide dismutase or a polypeptide analog thereof having substantially the same amino acid se quence as, and the biological activity of, naturally- occurring Cu-Zn superoxide dismutase from a composition which comprises cells containing Cu-Zn superoxide dismutase or a polypeptide analog thereof comprising:
• the Cu-Zn superoxide dismutase or polypeptide analog thereof may be human Cu-Zn superoxide dismutase or polypeptide analog thereof, for example, the non-acetylated polypeptide analog of human Cu-Zn superoxide dismutase.
• any eucaryotic cell which produces naturally-occurring Cu- Zn superoxide dismutase or polypeptide analogs thereof may be used.
• the eucaryotic cell may be, for example, a yeast cell or a mammalian cell.
• the method can also use a bacterial cell.
• the bacterial cell in which the Cu-Zn superoxide dismutase or polypeptide analog thereof is produced may be any bacterium in which a DNA sequence encoding the Cu-Zn superoxide dismutase or polypeptide analog thereof has been introduced by recombinant DNA techniques.
• the bacteria must be capable of expressing the DNA sequence and producing the desired protein.
• Presently preferred bacterial cells comprise cells of an Escherichia coli strain.
• the bacteria may be any strain including auxotrophic, prototrophic and lytic strains; F + and F- strains; strains harboring the cl 857 repressor sequence of the ⁇ prophage; and strains deleted for the deo repressors or the deo gene.
• Examples of wild type Escherichia coli strains which may be used are the prototrophic strain designated ATCC Accession No. 12435 and auxotrophic strain MC1061 (ATCC Accession No. 67361).
• Escherichia coli strains harboring the ⁇ cl 857 represser sequence which may be used are the auxotrophs A1645 containing plasmid pNd-SOD NN -12 (ATCC Accession No. 53166), A1637 containing plasmid pTV104(2) (ATCC Accession No. 39384), and A2097 containing plasmid pSOD ⁇ 2 (ATCC Accession No. 39786); and the prototrophs A4200 containing plasmid pHG44 (ATCC Accession No. 53218) and A4255 containing plasmid pSOD ⁇ MAX-12 (ATCC Accession No. 67177).
• Examples of lytic Escherichia coli strains which may be used include Escherichia coli strain A4048 containing plasmid pGH44 (ATCC Accession No. 53217).
• F- strains which may be used to express DNA encoding superoxide dismutase are Escherichia coli strain S ⁇ 930 (F-) containing plasmid pMF 5534 deposited under ATCC Accession No. 67703 and Escherichia coli strain W3110 (F-) containing plasmid pMFS 929 deposited under ATCC Accession No. 67705; strain W3110 (F-) was obtained from the Escherichia coli Genetic Stock Center (C.G.S.C.), Department of Biology, Yale University, P.O. Box 6664, New Haven, Connecticut, as C.G.S.C. strain No. 4474.
• C.G.S.C. Escherichia coli Genetic Stock Center
• Escherichia coli strains deleted for the deo gene or deo repressors which may be used include S ⁇ 732 containing plasmid pMF 2005 (ATCC Accession No. 67362), S( ⁇ 540 containing plasmid pJBF 5401 (ATCC Accession No. 67359) and S ⁇ 930 containing plasmid pEFF 920 (ATCC Accession No. 67706) (see European Patent Application Publication No. 0303972, published February 22, 1989).
• the bacterial cell may contain the DNA sequence encoding the superoxide dismutase analog in the body of a vector DNA molecule such as a plasmid.
• the vector or plasmid is constructed by recombinant DNA techniques, known to those skilled in the art, to have the sequence encoding the superoxide dismutase incorporated at a suitable position in the molecule.
• the plasmids used for production of superoxide dismutase can harbor a variety of promoters including but not limited to ⁇ promoter or deo promoters.
• plasmids which may be used for production of superoxide dismutase are the following:
• the treatment of the composition which comprises cells containing Cu-Zn superoxide dismutase or a polypeptide analog thereof so as to separate soluble proteins present in the cells from whole cells, cellular debris and insoluble proteins may comprise treating the composition so as to disrupt the cells and obtain a cellular extract therefrom, and then subjecting the resulting cellular extract to centrifugation so as to obtain the solution containing the soluble proteins.
• the cell walls of cultured bacterial cells may be disrupted by any of a number of methods known to those skilled in the art including, but not limited to, high shear mixing, sonication, mechanical disruption, explosion by pressure, osmotic shock, etc.
• the cells may secrete the Cu-Zn superoxide dismutase or polypeptide analog thereof into the medium, such that no cell disruption is required.
• the cells may then be removed by centrifugation or other means known in the art and the medium used directly for purification of the Cu-Zn superoxide dismutase or polypeptide analog thereof.
• the disrupted cell suspension (if disruption is required) may then be centrifuged or separated by other means known to those skilled in the art in order to recover a supernatant containing soluble Cu-Zn superoxide dismutase or polypeptide analog thereof.
• the superoxide dismutase solution may subsequently be heated and cooled.
• the solution containing soluble proteins including Cu- Zn superoxide dismutase or polypeptide analog thereof, is treated with a second solution containing a salt at a concentration such that the soluble proteins other than Cu-Zn superoxide dismutase or the polypeptide analog thereof present in the solution containing the soluble proteins are rendered capable of binding to an appropriate hydrophobic substance.
• the salt may be any suitable salt known in the art. Suitable salts which may be used include sodium sulphate and sodium chloride, although ammonium sulphate is preferred. Other salts which may be used include potassium chloride and ammonium acetate. Any combination of the foregoing salts may also be used.
• the final salt concentration depends on, and is characteristic of, the salt which is used.
• the final salt concentration varies from 1.2- 1.9M and is preferably 1.6M;
• the concentration range may vary between 0.9-1.25M, preferably 1.0M; and for sodium chloride the range is 3.0- 4.5M, preferably 4.0M.
• Optimal salt concentration ranges for other salts may be readily ascertained by those skilled in the art.
• the appropriate hydrophobic substance preferably comprises phenyl Sepharose.
• the appropriate hydrophobic substance may also comprise a suitable resin having a phenyl functional group, preferably a polysaccharide resin having phenyl groups present thereon.
• the contact with the hydrophobic substance preferably is effected by passing the solution containing the soluble proteins through a column containing the hydrophobic substance.
• Any hydrophobic column may be used, preferably phenyl-
• the functional group may be phenyl, benzyl, octyl, butyl and the matrix may be any of those discussed below.
• the soluble proteins other than Cu-Zn superoxide dismutase or the polypeptide analog thereof in the solution are bound to the hydrophobic substance in the column, thus separating the Cu-Zn superoxide dismutase or polypeptide analog thereof from the other soluble proteins.
• the resulting solution containing purified, enzymatically active Cu-Zn superoxide dismutase or polypeptide analog thereof is then recovered.
• the method described above for recovering a solution containing purified, enzymatically active Cu-Zn superoxide dismutase or a polypeptide analog thereof further comprises the following steps prior to the treatment with a second solution containing a salt:
• the resulting solution containing the soluble proteins after its separation from whole cells, cellular debris and insoluble proteins, is treated so as to obtain a more concentrated solution containing purified Cu-Zn superoxide dismutase or polypeptide analog thereof.
• the treatment of the solution to obtain a more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof comprises ultrafiltration.
• the ultrafiltration techniques are known to those skilled in the art.
• the exact filtration conditions, such as permeate flow rate and filter cut-off range are not critical and may be readily ascertained by a person skilled in the art of protein purification.
• Various systems which may be used include, but are not limited to, a PUF-100 Unit and a Pellicon Cassette System (Millipore).
• Ultrafiltration may be further conducted using a smaller molecular weight cut-off. It should be understood that the particular molecular weight cut-off used or the order used for various molecular weight cut-off filters is not critical to an understanding of the invention.
• the Cu-Zn superoxide dismutase present in the more concentrated solution may comprise a, b and c isoforms of Cu-Zn superoxide dismutase or polypeptide. analog thereof.
• the resulting more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof is then treated so as to produce three separate solutions, each of which has an increased concentration of one of either of the a, b or c isoform.
• the treatment of the concentrated solution comprises anion exchange chromatography.
• the then-resulting solution which has an increased concentration of the b isoform of purified Cu-Zn superoxide dismutase or polypeptide analog thereof is subjected to cation exchange chromatography so as to produce three further separate solutions, each of which has a further increased concentration of one of either of the a, b or c isoform.
• the then-resulting solution which has a further increased concentration of the b isoform of purified Cu-Zn superoxide dismutase or polypeptide analog thereof is then treated so as to further purify the Cu-Zn superoxide dismutase or polypeptide analog thereof contained therein prior to treating the resulting solution containing the further purified Cu-Zn superoxide dismutase or polypeptide analog thereof with a second solution containing a salt.
• the treatment of the then-resulting solution comprises strong anion exchange chromatography, preferably by passing the then- resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.
• a strong anion exchange column which may be used include Q-Sepharose and QAE Sephadex A-25.
• Functional groups may be any quaternary amino group such as quaternary amino ethyl and the matrix may be any of those discussed below.
• the then-resulting solutions which have an increased or further increased concentration of one of either of the a or c isoform may be treated so as to increase the concentration of b isoform and reduce the concentration of a isoform and c isoform of the polypeptide analog contained in each of the solutions prior to treating the then-resulting solution containing the further purified Cu-Zn superoxide dismutase or polypeptide analog thereof with a second solution containing a salt in step (b).
• the treatment of the then-resulting solutions prior to step (b) comprises:
• the treatment of the resulting combined solution so as to produce a solution which has an increased concentration of the b isoform comprises incubation.
• the solutions with an increased concentration of the b isoform from step (b) can further be treated so as to purify the b isoform present in the solutions.
• the treatment of the solution comprises ion exchange chromatography.
• the ion exchange chromatography may comprise anion exchange chromatography and further comprise cation exchange chromatography.
• the resulting solution containing purified, enzymatically active Cu- Zn superoxide dismutase or polypeptide analog thereof is recovered.
• the recovered resulting solution may then be treated so as to obtain a more concentrated solution containing purified Cu-Zn superoxide dismutase or polypeptide analog thereof.
• the treatment of the recovered resulting solution to obtain a more concentrated solution of purified Cu-Zn superoxide dismutase or polypeptide analog thereof comprises ultrafiltration.
• the resulting more concentrated solution containing purified Cu-Zn superoxide dismutase or polypeptide analog thereof may then be treated so as to obtain a solution containing more purified Cu-Zn superoxide dismutase or polypeptide analog thereof.
• the treatment of the resulting solution comprises strong anion exchange chromatography, prefer ably by passing the resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.
• the resulting solution containing purified, enzymatically active Cu-Zn superoxide dismutase or polypeptide analog thereof is recovered and treated so as to obtain a solution containing more purified Cu-Zn superoxide dismutase or polypeptide analog thereof.
• the treatment of the resulting solution comprises strong anion exchange chromatography, preferably by passing the resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.
• the resulting solution containing the soluble proteins after its separation from whole cells, cellular debris and insoluble proteins, is treated so as to purify the Cu-Zn superoxide dismutase or polypeptide analog thereof contained therein prior to treating the resulting solution containing the soluble proteins with a second solution containing a salt.
• the treatment of the resulting solution comprises strong anion exchange chromatography, preferably by passing the resulting solution through a column of a polysaccharide resin having quaternary amino functional groups present thereon.
• the method described above for recovering a solution containing purified, enzymatically active Cu-Zn superoxide dismutase or a polypeptide analog thereof further comprises treating the resulting solution containing the soluble proteins, after its separation from whole cells, cellular debris, and insoluble proteins (the soluble proteins including Cu-Zn superoxide dismutase or polypeptide analog thereof comprising a, b and c isoforms of Cu-Zn superoxide dismutase or polypeptide analog thereof) so as to increase the concentration of b isoform and reduce the concentration of a isoform and c isoform of the polypeptide analog contained in the solution prior to treating the resulting solution containing the soluble proteins with a second solution containing a salt.
• the treatment of the resulting solution containing the soluble proteins prior to treatment with a second solution containing a salt comprises:
• the treatment of the resulting combined solution so as to produce a solution which has an increased concentration of the b isoform comprises incubation.
• the treatment of the resulting solution so as to produce three separate solutions preferably comprises ion exchange chromatography.
• any number of ion exchange columns may be used including weak anion exchange, cation exchange or so-called "strong anion” exchange columns.
• the protein purification using ion exchange chromatography may involve applying a high concentration of solution through an anion exchange followed by a cation exchange, or a cation exchange followed by an anion exchange or an anion exchange followed by an anion exchange or a cation exchange followed by a cation exchange.
• only one exchange, i.e., either cation or anion, may be required.
• the anion exchange of this method and the method disclosed above may involve, for example, a DEAE Sepharose fast-flow column and the cation exchange may involve, for example, a CM Sephadex or S-Sepharose column.
• Weak anion exchange columns used usually have a tertiary amine, e.g., diaminoethyl, as a functional group, although amino ethyl may also be used.
• the matrix may be based on inorganic compounds, synthetic resins, polysaccharides or organic polymers. Examples of matrices which may be used include agarose, cellulose, trisacryl, dextran, glass beads, oxirane acrylic beads, acrylamide, agarose/polyacrylamide copolymer (Ultragel) and hydrophobic vinyl polymer (Fractogel).
• CM Sepharose fast flow examples include CM Sephadex or S Sepharose columns.
• Functional groups which may be used include carboxymethyl, phospho groups or sulphonic groups such as sulphopropyl. Any of the matrices discussed above for weak anion exchange columns may be used in cation exchange columns as well.
• the solution with an increased concentration of the b isoform which is recovered can further be treated so as to purify the b isoform present in the solution.
• the treatment of the solution comprises ion exchange chromatography.
• the ion exchange chromatography may comprise anion exchange chromatography, and may further comprise cation exchange chromatography.
• the contaminating Escherichia coli or bacterial proteins and endotoxins are reduced by about one-thousand fold.
• the critical step leading to reduction in contaminating ECP is the hydrophobic separation step following the optional anion and cation exchange chromatography steps.
• This hydrophobic separation step is novel. In this hydrophobic separation step, it is the desired superoxide dismutase protein which passes through the hydrophobic column while undesirable Escherichia coli proteins are absorbed.
• the use of such a step in the purification procedure surprisingly and unexpectedly leads to a marked decrease in the level of contamining Escherichia coli proteins.
• the invention also provides a novel method involving subunit exchange for increasing the yield of recovered solutions having an increased concentration of b isoform of an enzymatically-active polypeptide analog of Cu-Zn superoxide dismutase from a composition which comprises cells containing a, b and c isoforms of the polypeptide analog which comprises:
• peaks containing predominantly a and c isoforms are combined and incubated to form b isoforms which may then be used for further purification downstream.
• the treatment of the resulting combined solution so as to produce a solution which has an increased concentration of the b isoform comprises incubation.
• the polypeptide analog of Cu-Zn superoxide dismutase may be a polypeptide analog of human Cu-Zn superoxide dismutase.
• the treatment of the composition so as to separate soluble proteins present in the cells from whole cells, cellular debris and insoluble proteins comprises treating the composition so as to disrupt the cells and obtain a cellular extract therefrom, and then subjecting the resulting cellular extract to centrifugation so as to obtain the solution containing the soluble proteins.
• the treatment of the resulting solution containing the soluble proteins so as to produce three separate solutions comprises ion exchange chromatography.
• the method further comprises treating the solution with an increased concentration of the b isoform from step (c) or (f) so as to purify the b isoform present in the solution.
• the treatment of the solution comprises ion exchange chromatography.
• the ion exchange chromatography may comprise anion exchange chromatography and may further comprise cation exchange chromatography.
• the plasmid used for production of hSOD analog is plasmid pSOD ⁇ 1 Tll ( Figure 1).
• Plasmid pSOD ⁇ 1 T11 is a high level expressor of hSOD analog protein under the control of the strong leftward promoter of bacteriophage ⁇ (P L ) which is thermoin- ducibly controlled by the CI857 temperature sensitive repressor situated on the host chromosome.
• Plasmid pSOD ⁇ 1 T11 was used to transform prototrophic Escherichia coli strain A4255, and the resulting bacterium is used for production of hSOD analog.
• Prototrophic Escherichia coli strain A4255 was prepared from prototrophic Escherichia coli strain ATCC Accession No. 12435 (obtained from the ATCC collection) by transfection with Tn10 transposon carrying defective ⁇ prophage (CI857 ⁇ H1 ⁇ Bam N + ) and subsequently deleted for tetracycline resistance gene (carried by Tn10).
• Escherichia coli strain A4255 containing plasmid pSOD ⁇ 1 T11 was deposited in the ATCC under Accession No. 53468.
• the bacteria producing hSOD analog (Escherichia coli strain No. A4255/pSOD ⁇ 1 T11) are cultivated overnight at 30°C in shake flasks containing CAS medium, which is of the following composition:
• Deionized water 450 ml
• the cell suspension is dispensed in 2 ml aliquots into sterile, plastic screw-top vials (Nunc Cryo-tubes, cat.
• the inoculum is propagated in 20 g/L casein hydroly- sate, 10 g/L yeast extract, 5 g/L NaCl, and 12.5 mg/L tetracycline HCl.
• Sterile medium in shaker flasks is inoculated from stock culture and incubated 8-10 hours on a shaker at 30°C at approximately 240 rpm. The flask contents are used to inoculate the seed fermentor.
• the seed fermentor medium is identical to production medium (see below) except that all the glucose (30 g/L) is added at the start of fermentation; the medium lacks added copper and zinc.
• Sterile medium is inoculated with culture, and incubated 15 hours at 30°C, pH 7 + 0.1 with agitation and aeration to maintain a dissolved oxygen level of about 20% saturation. Contents of the seed fermentor are then transferred to the production fermentor.
• the production medium contains: Casein hydrolysate 20 g/L
• the cake obtained as described above is resuspended in 25 mM potassium phosphate buffer, pH 7.8, containing 155 mM NaCl.
• the composition of the buffer is:
• Harvested cell cakes are weighed and stored frozen at -20°C. Cakes weighing 50-60 kg from one or more fermentations are processed downstream.
• the wet cell cake (51.6 kg containing 260 gr hSOD analog) is thawed at 4-10°C and suspended in 4 liters of 25 mM Tris-75 mM NaCl buffer pH 7.8 for each kilogram of wet cells (total volume of suspension is 258 liters).
• a Polytron high-shear mixer (Kinematica, Luzern) aids in dispersing the cake.
• the pH of the suspension is adjusted to 7.3 - 7.5 if necessary.
• the suspended cells are then disrupted by any suitable method, e.g., by sonication or mechanical disruption. The following method is preferred.
• the cells are fed into a Dynomill KD-5 bead mill disrupter (Willy A.
• the bead mill is circulated with a cooling liquid at -20°C to maintain the temperature of the bead mill discharge at 8-25°C.
• the bead mill discharge is fed again into the Dynomill and grinding of the suspension is repeated according to the above conditions.
• the pH of the disrupted cell suspension is adjusted to 7.3 - 7.5 if necessary.
• the suspension is centrifuged on the Cepa 101 tubular bowl centrifuge (Carl Padberg, Lahr/Schwarzwald) at 14,000 rpm (16,000 G's).
• the feed rate to the centrifuge is 45-55 L/hr.
• the cake (weighing 23.4 kg) is discarded.
• the supernatant (243 L) contains the soluble hSOD analog and is saved.
• the supernatant from the previous step (243 L containing 250 gr hSOD) is supplemented with 0.25 N NaCl by adding 5 N NaCl providing a final concentration of 0.325 N NaCl and the pH is adjusted to 7.3-7.5 if necessary.
• the solution is transferred into a jacketed bioreactor (Bioengineering AG., Wald, Switzerland).
• An ICI silco- lapse 5000 antifoam is added to a final concentration of 10-15 ppm active material.
• the temperature is raised at a slow rate to 65°C while stirring gently to avoid foaming.
• the heat treatment at 65°C is carried out for 2 hours and terminated by cooling the solution in the bioreactor to 40°C and then transferred to a cold room for incubation at 4°C overnight (10-16 hours) without stirring.
• the cooled solution is centrifuged on the Cepa-101 at 40-60 L/hr. All other centrifugation conditions are identical to those of cell disruption.
• the cake (weighing 11.3 kg) is discarded.
• the clarified supernatant (233 L) containing the soluble hSOD analog is saved and the pH is adjusted to 7.3-7.5.
• Any ultrafiltrate method with a cut-off point at 100K may be used in this step.
• Examples of such methods are the Pellicon cassette system or Amicon's hollow fiber system.
• the preferred ultrafiltration method is the
• Millipore PUF-100 Unit Process Ultrafiltration System equipped with two 50 ft 2 spiral-wound membrane cartridges with 100,000 molecular weight cut-off ratings (Millipore Intertech, Inc., Bedford, Massachusetts). This is used to concentrate the protein solution of the previous step (230 L containing 232 gr hSOD) to about 10% of the initial volume (20 L) and the filtrate (210 L containing some of the hSOD) is saved. The initial permeate rate is 6 L/min. The retentate containing the rest of the hSOD is diafiltered by continuous addition of fresh 20 mM Tris buffer at pH 7.8 to the well-mixed retentate at a rate just equal to the permeate rate.
• the volume of retentate remains constant while the hSOD passes through the membrane. Diafiltration is continued until 2-3 times the volume of the clarified protein solution used in the beginning of this step is collected (460 liters of filtrate). At this point the permeate OD is checked and found to be in the range 0.1-0.2. [During this period, the membranes are depolarized several times whenever a decrease in permeate flow rate is observed (below 4 L/min). This is achieved by diluting the retentate to 40 liters and recirculating for a few minutes with the permeate valves shut off. The retentate is concentrated again to 20 liters and diafiltration resumes.] The filtrate is collected, combined with the permeate (210 L) of the concentration step (total of 670 L) and the retentate is discarded.
• the feed, retentate and permeate are all examined by Superose 6B gel chromatography in order to determine the extent and quality of the separation.
• the conditions of the gel column are:
• the 100 K molecular weight cut off fractionation of hSOD analog has been practiced by ultrafiltration with both the PUF-100 Unit and the Pellicon Cassette System.
• the Pellicon System is suitable for smaller batches of hSOD, as described below.
• the cell cake weight of this batch was 9.25 Kg containing 45 gr hSOD analog.
• the clarified protein solution obtained at the end of the heat treatment step (44 liters containing 41.6 gr hSOD) is ultrafiltered through two Pellicon Cassette
• Any ultrafiltration method with a cut-off point of 10 K may be used in this step.
• the preferred method is described here, where a Millipore PUF-100 unit and a Pellicon Cassette System (10 K cut-off) are used sequentially.
• the combined permeate from the previous step (670 L containing 229 gr hSOD) is inline fed into a Millipore
• the hSOD analog is concentrated to a maximum of 100 gr/L (10 L containing 229 gr).
• the retentate is drained out of the Pellicon and the ultrafilter is washed with a small amount of 20 mM Tris pH 7.8 which is combined with the retentate.
• the combined retentate volume after addition of the wash is divided into portions containing an amount equivalent to about 75 gr hSOD analog each (3 portions) which are processed directly or stored at -20°C. Prior to proceeding to the next purification step, frozen hSOD analog solutions are thawed at 37°C.
• the retentate and permeate are examined by Superose 6B gel chromatography to determine the extent and quality of the separation.
• the conditions of the gel column are:
• Any weak anion exchange (e.g. tertiary amine) method can be used in this step but DEAE Sepharose fast-flow chromatography is preferred.
• a retentate solution from the previous step (3300 ml of 23 mg hSOD analog/ml) is diluted to 10 mg hSOD/ml with 20 mM Tris, pH 7.8.
• the solution is loaded onto a KS- 370 stack column [37 cm x 15 cm (Pharmacia, Uppsala)], packed with 16 L of DEAE Sepharose C1-6B fast flow anion exchanger at a maximum linear flow velocity of 164 cm/hr (180 L/hr).
• An equivalent of 75 gr hSOD analog maximum is loaded onto 16 liters of resin without peak separation interference.
• pyrogen-free water purified by reverse osmosis system (Hydro-Hammer, Tel Aviv) is used.
• the elution flow velocity is up to 180 L/hr and the hSOD analog peak is collected in 0.8 to 1.5 bed volumes (21 liters containing 50 gr hSOD) and is saved.
• the average concentration of the hSOD analog in the pool is approximately 2.5 gr hSOD/L.
• Concentration after DEAE The eluted peaks from one or more loadings are concentrated by ultrafiltration through a Millipore Pellicon Cassette System equipped with three 10,000 molecular weight cut-off cassettes (type PTGC) of 5 ft 2 each until the calculated activity in the retentate is up to 100 mg hSOD/ml.
• the ultrafiltrate should contain no hSOD analog and is discarded.
• the retentate is usually concentrated by a factor of roughly 20 (2 liters of retentate).
• the retentate containing the hSOD analog is drained from the Pellicon, and the ultrafilter is washed with a small amount of 20 mM Tris pH 7.8 which is combined with the retentate.
• the combined retentate volume after addition of the wash is divided into portions each containing not more than an equivalent of 75 gr hSOD analog and that is processed directly or stored at -20°C. Prior to proceeding to the next purification step, frozen hSOD solutions are always thawed at 37°C.
• CM Sephadex or S-Sepharose.
• S-Sepharose any cation exchange method can be used, e.g. CM Sephadex or S-Sepharose.
• the preferred method is carboxymethyl (CM) Sepharose Fast Flow (FF).
• a retentate solution from the previous step (4000 ml containing 21.15 mg hSOD analog/ml) is dialyzed on the day of loading onto the CM column.
• the dialysis is carried out by continuously adding 40 mM sodium acetate at pH 7.8 to the retentate using the Millipore Pellicon Cassette System. All other concentration and dialysis conditions are identical to those described in the DEAE Chromatography step.
• the dialysis is completed when the filtrate conductivity is equal to that of the dialysis buffer (20-25 volumes per volume of retentate). The permeate is discarded.
• the retentate is drained out of the Pellicon and the ultra- filter is washed with a small amount of 40 mM sodium acetate pH 7.8 which is combined with the retentate.
• the progress of the run is monitored by continuously following the absorbance of the eluate at 280 nm, and the elution profile of this column is shown in Figure 2B.
• the column is washed at the above flow rate with three to four bed volumes (48-64 L) of 40 mM sodium acetate adjusted to pH 4.8 with glacial acetic acid.
• the eluate from the loading and washing steps is discarded (however, see Example 4).
• a step change in eluent to 85 mM sodium acetate adjusted to pH 4.8 with glacial acetic acid displaces the hSOD analog off the column; this is the peak shaded in black in Figure 2B.
• the maximum elution flow velocity is 180 L/hr and the hSOD analog peak is collected in 0.75 to 1.25 bed volume (12 L) and is saved.
• the protein solution is titrated immediately to pH 7.8 with 1 N NaOH.
• Concentration and dialysis after CM The eluted peaks from one or more loadings carried out on the same day are concentrated up to 100 mg hSOD analog/ml. The concentration conditions are identical to those described in the DEAE Chromatography step. The retentate is then dialyzed against 50 mM Tris pH 7.8 using the Millipore Pellicon Cassette System. The dialfiltration is performed until the permeate conductivity is equal to that of the dialysis buffer (at least 20 volumes of dialysis buffer per one volume of retentate are required).
• the retentate containing the dialyzed hSOD analog is drained from the Pellicon and the ultrafilter is washed with a small amount of 50 mM Tris pH 7.8 which is combined with the retentate.
• the combined retentate is divided into portions of 150-160 gr hSOD analog, each of which are processed directly or stored at -20°C.
• phenyl-Sepharose chromatography is used to achieve hydrophobic separation.
• the hSOD analog which is applied in overcapacity and in high salt concentration, does not attach to the column but goes straight through; this achieves purification of hSOD analog from residual Escherichia coli protein (ECP) and endotoxin which are absorbed.
• ECP Escherichia coli protein
• This novel step achieves a surprising degree of reduction of ECP and endotoxin contamination.
• the ECP levels are reduced by about 3 orders of magnitude; the measurement of ECP by a solid- phase immunoradiometric assay is described in Example 5 where the reduction of ECP by means of the phenyl-Sepharose column is detailed (Table 2).
• the endotoxin level is reduced by a similar extent, from about 10,000 ppm to less than 10 ppm, as measured by the Limulus Amebocyte Lysate (LAL) assay described in U.S. Pharmacopeia (U.S.P.) XXI 1165-1166 (1985).
• LAL Limulus Amebocyte Lysate
• a retentate solution from the previous step (5000 ml containing 31.6 mg hSOD analog/ml), is brought by 50 mM Tris pH 7.8 to a protein concentration of 25-30 mg hSOD/ml.
• a buffer containing 50 mM Tris - 3.2 M ammonium sulfate at pH 7.8 is used to dilute volume per volume the proteinaceous solution to a final concentration of 12.5 to 15 mg hSOD analog/ml (15 mg hSOD/ml is preferable) in 1.6 M ammonium sulfate and 50 mM Tris at pH 7.8.
• the solution is stirred gently for 30-60 minutes at room temperature and then loaded on a 11.3 x 30 cm column (113 Bioprocess, Pharmacia, Uppsala) packed with 3 L of phenyl-Sepharose fast flow at a linear flow velocity of 3-6 L/hr.
• a maximum of 150 gr hSOD analog can be loaded on the 11.3 x 30 cm column.
• the column is eluted by 50 mM Tris-1.6 M ammonium sul fate, pH 7.8 which displaces the hSOD analog off the column.
• the elution flow velocity is 3-6 L/hr.
• the progress of the run is monitored continuously by following the absorbance of the eluate at 280 nm.
• the eluted hSOD analog peak has maximal absorbance after about one and one-half bed volume of eluant. Material eluted at the beginning of the peak (up to 2% of the maximum absorbance) is discarded. The major portion of the peak is saved (from where the absorbance is 2% of the maximum absorbance at the beginning of the peak to 5% of the maximum at the end (9-12L)).
• the eluted peak is concentrated by ultrafiltration through a Millipore Pellicon Cassette System equipped with three 3000 molecular weight cut-off cassettes of 5 ft 2 each until the calculated activity in the retentate is up to 25 mg hSOD analog/ml.
• the ultrafiltrate should contain no hSOD and is discarded.
• the retentate is then dialyzed against 20 mM Tris pH 8.8. The dialysis is completed when the filtrate pH and conductivity are equal to that of the dialysis buffer (at least 20 volumes per volume of retentate are needed).
• the retentate containing the dialyzed hSOD analog is drained from the Pellicon and the ultrafilter is washed with a small amount of 10 mM ammonium bicarbonate, pH 8.0 which is combined with the retentate. All mentioned ultrafiltration activities are performed in a cold room (4°-10°C). The combined retentate (5460 ml containing23.9 hSOD/ml) can be transferred to the next step or stored frozen at -20°C.
• a strong anion exchanger is used.
• This can be, for example, QAE Sephadex A-25, but the preferred method is to use Q-Sepharose (QS).
• QS Q-Sepharose
• Steps G and H can be interchanged, but the order given here is preferred. It is also possible for the superoxide dismutase analog solution to be of sufficient purity after the hydrophobic column exchange step that this step may be unnecessary.
• the retentate solution from the previous step (3.88 L of 28.6 mg hSOD analog/ml) is diluted to 5 mg hSOD analog/ml with 20 mM Tris, pH 8.8 and supplemented with 20 mM NaCl.
• the solution is stirred gently at 20-30°C for 30-45 minutes and loaded on a KS 370 stack column (37 x 15 cm, Pharmacia, Uppsala) packed with 16 L of Q- Sepharose fast flow anion exchanger, at a linear flow velocity of 90-180 L/hr.
• a maximum of 200 gr hSOD analog can be loaded on the KS 370 column.
• the column is washed with one to two bed volumes of 20 mM Tris-45 mM NaCl, pH 8.8 and the eluate is discarded.
• a step change in eluent to 20 mM Tris-130 mM NaCl, pH 8.8 displaces the hSOD analog off the column.
• the elution flow velocity is 90-180 L/hr.
• the progress of the run is monitored continuously by following the absorbance of the eluate at 280 nm/
• the eluted hSOD analog peak has maximal absorbance after about one bed volume of eluent.
• the major portion of the peak (50-60 liters) is saved. Material eluted at the end of the peak (where the absorbance is lower than 5% of the maximal absorbance) is discarded.
• Concentration and dialysis after QS The eluted peak is concentrated to an OD of up to 45 then dialyzed against 10 mM ammonium bicarbonate pH 8.0 using the Millipore Pellicon Cassette System in a cold room (5°C). The dialysis is completed when the filtrate pH and conductivity are equal to that of the dialysis buffer (at least 20 volumes per volume of retentate are needed). All other concentration and dialysis conditions are identical to those described in the DEAE and the CM Chromatography steps.
• the retentate containing the dialyzed hSOD analog is drained from the Pellicon and the ultrafilter is washed with a small amount of 10 mM ammonium bicarbonate, pH 8.0 which is combined with the retentate.
• the combined retentate wash (2.34 L of 44.45 mg hSOD analog/ml) is transferred to the next step.
• Freeze drying of the bulk hSOD analog is carried out as follows: The hSOD analog solution is diluted using 10 mM ammonium bicarbonate, pH 8.0 to a final concentration of 7 to 10 mg hSOD/ml (10.4 L of 10 mg hSOD/ml) and then divided into approximately 600 ml fractions under aseptic conditions in a laminar flow hood (Contamination Control Inc., Lansdale, Pennsylvania). Each portion is added to a 2 liter glass jar preheated for 4 hours in a 180°C oven. The jars are frozen quickly using an acetone-dry ice mixture and put on a freeze dryer (Freezemobile-24, Virtis Company, New York). The outer surface of the jars is exposed to ambient temperature for 60-120 hours throughout the drying cycle until the hSOD analog is dry, and it is then packaged.
• the overall yield of hSOD analog is about 25% and it is extremely pure, containing less than 10 ng ECP per mg hSOD analog.
• Cu-Zn SOD enzymes from different species consist of dimers built from two chemically identical subunits and yet are known to appear in several discrete forms on isoelectric focusing gel (IEF).
• IEF isoelectric focusing gel
• the dimeric human Cu-Zn SOD can be separated into three major electroforms.
• the electroforms may represent post-translation modifications.
• the IEF and Mono-Q patterns of natural Cu-Zn hSOD can be explained in terms of there being two types of subunits, which can result in one or two extra negative charges per dimer. These differences in charge provide the different pi values.
• the subunits designated x and y could associate to yield xx, xy and yy dimers which would correspond to the observed three major dimeric isoforms, designated a, b, and c, respectively.
• the recombinant hSOD analog also consists of three discrete isoforms with pis of 5.16, 4.95, and 4.85 that are the a, b and c isoforms, respectively.
• the Cu-Zn hSOD that has been extracted from red blood cells also has three isoforms with pis of 4.85, 4.75, and 4.65 i.e., natural hSOD is more acidic than the recombinant Cu-Zn hSOD analog.
• the acidic shift appearing in the IEF pattern of the authentic hSOD versus the recombinant analog can be explained by the net decrease of two positive charges at the N-terminal residues of the authentic Cu- Zn hSOD which are blocked by one N-acetyl group per polypeptide chain (1, 6, 10).
• each sample is first concentrated by ultrafiltration, as described in Example 3 step E, and then dialyzed against 20 mM tris pH 7.8. The four samples are then combined.
• Incubation of the combined samples is performed under conditions that can be varied e.g., 4 hours at 37°C, or 2 hours at 37°C followed by overnight at room temperature, or 30 minutes at 50°C. All three sets of incubation conditions have a fairly similar effect on the formation of the intermediate isoform b by means of subunit exchange from isoforms a and c. The kinetics of formation of the new b isoform is temperature dependent.
• the amount of the new b dimer formed is, of course, dependent on the relative amounts of a and c dimers added to the incubation mixture. Thus, the proportion in which the four samples are mixed is critical for. the overall success of the exchange process. vi. Following the incubation the mixture becomes b- rich ( Figure 6). At the end of the exchange step, the material is chromatographed on DEAE- Sepharose and on CM-Sepharose (i.e. steps E and F of Example 3 are repeated); this re-chromatography is shown in Figures 7A and B. This purified preparation is then added to the bulk preparation held at step F(iii). steps G, H and I are then performed on the combined material as one batch.
• ECP Escherichia coli Protein
• This assay is to quantify residual ECP presence in the purified recombinant hSOD analog preparation.
• IRMA immunoradiometric assay
• the ECP standard was prepared from a mock purification process (Example 3) of Escherichia coli reference (Escherichia coli containing the plasmid without the gene coding for hSOD analog); the fraction used was that which eluted from the CM-Sepharose column. Antibody to this ECP was raised in rabbits and purified IgG's to the ECP were obtained.
• the solid-phase IRMA is accomplished by first coating the wells of a plastic 96-well plate with purified IgG's against the mock-purified, CM-eluted ECP fraction. CM-purified ECP, at serial dilutions, is allowed to bind to the IgG in the wells. Subsequently, 125 I- labeled anti-ECP antibody is added, thereby providing a
• the IRMA proved to be sensitive to 1 ng/ml of ECP, which is significantly different from the zero point, as defined, by the minimum detectable dose method of Rodbard et al., Radioimmunoassay And Related Procedures In Medicine I (International Atomic Energy Agency, Vienna) p. 469, (1977).
• the above assay was used to monitor the ECP in various steps of hSOD analog purification.
• the antigen used was produced at Step F after the CM column. (See Example 3, Scheme I.)
• Table 2 the two subsequent steps G and H (phenyl-Sepharose chromatography and Q-Sepharose chromatography) remove efficiently virtually all ECP contaminants from the SOD analog preparation. Note that here steps G and H are reversed. This is a possible alternative order, as described in Example 3.
• a homogenized human liver preparation was heat-treated at 65°C and then centrifuged. Ammonium sulphate was added to the supernatant solution and the 65%-80% ammonium sulphate precipitate was obtained. A solution of this precipitate was applied to a DEAE ion-exchange column to separate the manganese superoxide dismutase (MnSOD) from the Cu-Zn superoxide dismutase (Cu-Zn SOD).
• MnSOD manganese superoxide dismutase
• Cu-Zn SOD Cu-Zn superoxide dismutase
• the resulting fractions which eluted in high salt concentration contained highly purified naturally-occurring Cu-Zn superoxide dismutase.
• the contaminant eucaryotic proteins remained bound to the column until eluted with water.

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